Many conditions and diseases are believed to be associated with, induced, and/or mediated by the amyloid-β peptide (“Aβ” or “amyloid-β”), a proteolytic fragment of the integral membrane glycoprotein, amyloid-β precursor protein (APP) [Kang et al., Nature, vol. 325, pp. 733-736 (1987)]. Examples of such diseases, conditions and/or cancers include progressive neurodegenerative disease, such as Alzheimer's disease (“AD”) or related Aβ-mediated dementia, and certain cancers, such as breast and endometrium cancers [see He et al., J. Biol. Chem., 274(21), pp 15014-15019(1999)].
Aβ has been identified as a 39-43 amino acid peptide having a molecular weight of about 4.2 kD, which peptide is substantially homologous to the form of the protein described by Glenner, et al., Biochem. Biophys. Res. Commun., 120:885-890 (1984), including mutations and post-translational modifications of the normal .beta.-amyloid peptide. Aβ peptide has been described in U.S. Pat. No. 6,262,302 as an approximately 39-43 amino acid fragment of a large membrane-spanning glycoprotein, referred to as the Aβ precursor protein (APP).
Aβ may interact with certain intracellular proteins and that interaction could lead to cytotoxic events. Examples of intracellular proteins believed to interact with Aβ are Endoplasmic Reticulum-associated Amyloid-β-peptide binding protein (“ERAB”) and L-3-Hydroxyacyl-CoA Dehydrogenase Type II (“HADH2”).
As used herein, “ERAB” refers to Endoplasmic Reticulum-associated Amyloid-β-peptide binding protein. ERAB has been identified as a dehydrogenase enzyme capable of binding Aβ [Yan et al., Nature, Vol. 389, pp. 689-95 (1997)]. As used herein, “HADH2” refers to L-3-Hydroxyacyl-Co Dehydrogenase Type II. HADH2, believed to be identical to ERAB, has been independently identified as a new L-3-hydroxyacyl-CoA dehydrogenase with an apparent role in the mitochondrial fatty acid β-oxidation pathway [He et al., J. Bio. Chem. Vol. 273. No. 17, pp. 10741-10746 (1998)]. The terms “ERAB”, “HADH2” and “HADH” are interchangeably used in the art to indicate the amyloid-β binding protein. Throughout the application, the term “ERAB or HADH2” is used to indicate the ERAB and HADH2 protein, as well as the ERAB and HADH2 gene.
ERAB or HADH2 is an NAD+ dependent dehydrogenase which catalyzes the reversible oxidation of L-3-hydroxyacyl-coA. The human short chain L-3-hydroxyacyl-CoA dehydrogenase gene is organized into six exons and five introns and maps to chromosome Xp11.2 [He et al., J. Biol. Chem, Vol. 273, pp. 10741-6 (1998)]. Sequence comparisons show that ERAB or HADH2 belongs to the short-chain dehydrogenase/reductase (“SDR”) family of enzymes. ERAB or HADH2 has been cloned, expressed, purified, and characterized from human brain [He et al., J. Biol. Chem., Vol. 273, pp. 10741-6 (1998)]. ERAB or HADH2 messenger RNA is expressed ubiquitously in normal human tissues. It is highest in liver and heart but ERAB or HADH2 is also expressed in normal brain.
Experimental evidence suggests that ERAB or HADH2 interacts with the Aβ peptide and can mediate its cytotoxicity. For example, ERAB or HADH2, normally found in the endoplasmic reticulum and mitochondria, has been shown to become redistributed to the plasma membrane fraction of cells in the presence of Aβ peptide [Yan et al., Nature, vol. 389, pp. 689-95 (1997)]. Likewise, it has been shown that the cytotoxic effects of Aβ on neuroblastoma cells in culture can be blocked by anti-ERAB or anti-HADH2 antibodies. Cells that overexpress ERAB or HADH2 and Aβ show elevated markers of cytotoxicity and cell stress compared to mock transfected controls; conversely, cells overexpressing catalytically inactive mutants of ERAB or HADH2 were no more insensitive than controls which overexpressed Aβ alone [Yan et al., J. Biol. Chem., vol. 274, pp. 2145-56 (1999)]. Further, the interaction of Aβ and ERAB or HADH2 links oxidoreductase activity with both apoptosis and amyloid toxicity [Spermann et al., FEBS Lett, 451(3), pp. 238-242 (1999)]. Thus, ERAB or HADH2 appears to mediate the intraneuronal toxicity of Aβ by acting on inappropriate substrates, possibly generating toxic aldehydes [Yan et al., J. Biol. Chem., vol. 274, pp. 2145-56 (1999)].
Alzheimer's disease (“AD”) is a progressive neurodegenerative disease of the brain resulting in diminished cognitive abilities, dementia, and ultimately death. AD can be diagnosed by a trained clinician through, for example, the patient history, physical examination, tests that measure memory and language skills, genetic testing, and magnetic resonance imaging (MRI).
A strong link has been established between the development of AD and the accumulation of “Aβ” outside of nerve cells in the brain [Storey et al., Neuropathology And Applied Neurobiology, vol. 25, pp. 81-97 (1999); Selkoe, Annual Review of Neuroscience, vol. 17, pp. 489-517 (1994); Small et al., Journal of Neurochemistry, vol. 73, pp. 443-9 (1999)]. Aβ is also the principal component of the extracellular plaques that are diagnostic of AD and species of the peptide have been shown to be engaged by intracellular targets [Yan et al., Nature, vol. 389, pp. 689-95 (1997)]. Aggregated Aβ appears to be toxic to neuronal cells in culture. Aβ has been reported to cause apoptotic (neuronal) cell death in vitro through the generation of nitric oxide and other free radicals. Aβ has also been reported as accumulating to form plaques both inside and outside nerve cells [Wilson et al., Journal of Neuropathology And Experimental Neurology, vol. 58, pp. 787-94 (1999)]. These plaques are believed to be strongly associated with the dementia caused by AD. There are several different ways that these plaques can damage the brain. One way they can cause damage is by disrupting the calcium channels. They can also create free radicals, which then damage the brain. When the plaques form between the nerve cells in the brain, microglia, a type of immune cell, can cause an inflammation leading to even more neurological damage.
In a normal brain, ERAB or HADH2 antigen is present at low levels, being predominantly localized in neurons. However, in neurons affected in AD, ERAB or HADH2 is found to be overexpressed relative to non-AD age matched controls, especially near deposits of Aβ [Yan et al., Nature, vol. 389, pp. 689-95 (1997)]. It has also been suggested that ERAB or HADH2 contributes to Aβ-associated pathogenesis of AD by reducing neuroprotective estrogen levels in the brain, based on the finding that the enzyme can also utilize estrogen as a substrate [Yan et al., J. Biol. Chem., vol. 274, pp. 2145-56 (1999); He et al., J. Biol. Chem., vol. 274, pp. 15014-9 (1998)].
Accordingly, compounds and compositions that modulate or inhibit ERAB or HADH2 activity find therapeutic utility in the treatment of ERAB or HADH2 mediated conditions and diseases. In addition to any therapeutic application, such ERAB or HADH2 inhibitors or modulators are useful in delineating the role of the ERAB or HADH2 enzyme in both normal cellular function and in Aβ pathogenesis.
Various pyrazole or pyrimidine derivatives have been reported for their pharmacological activity. For example, European Patent Publication Nos. EP 0 463 756 A1 and EP 0 526 004 A1, and U.S. Pat. Nos. 5,272,147 and 5,426,107 diclose certain pyrazol-[4,3-d]pyrimidine-7-one compounds that are reported to be selective cGMP PDE inhibitors. International publication Nos. WO96/16644, WO94/28902 and WO98/49166 disclose use of certain pyrazolo[4,3-d]pyrimidine-7-one compounds in treatment of impotence. U.S. Pat. No. 6,207,829 discloses a method for producing certain pyrazolo[4,3-d]pyrimidine-7-one and its intermediates. U.S. Pat. No. 6,197,774 reports certain pyrimidine derivatives that inhibit the formation of nitrogen monoxide, and their use in treatment of allergic diseases. U.S. Pat. No. 6,194,410 describes certain pyrazolopyrimidines and pyrazolotriazines having a sulphanyl group, that are reported to show selective affinity to 5HT-6 receptors and as being suitable for use in the treatment of central nervous disorders such as psychoses or schizophrenia. U.S. Pat. No. 4,666,908 discloses certain 5-substituted pyrazolo[4,3-d]pyrimidine-7-one compounds. U.S. Pat. Nos. 5,047,404, 5,707,998, and 5,294,611 describe certain fused pyrimidine derivatives, and quinazoline and quinazolinone compounds, respectively. U.S. Pat. Nos. 5,294,612, 5,656,629 and 5,541,187 disclose certain pyrazolo[4,3-d]pyrimidin-4-one compounds having substituents at the 6-position for treating cardiovascular diseases. U.S. Pat. No. 3,165,520 discloses certain coronary dilating pyrazolo-[3,4-d]-pyrimidine compounds.
WO 00/76969 reports a method of treating AD using certain isoindoline derivatives. WO 00/76987 and 00/76988 report a method of treating AD using certain thiazolidine derivatives.
WO 98/40484 discloses an isolated nucleic acid encoding an ERAB or HADH2, and a method for treating a neurodegenerative condition by administering an ERAB or HADH2 inhibiting agent in an amount effective to inhibit ERAB or HADH2 polypeptide binding to Aβ. WO 99/18987 discloses an isolated peptide of V-domain of a receptor for advance glycation end product (RAGE) and its use for inhibiting the interaction of Aβ with the RAGE to treat degeneration of a neuronal cell. WO 01/12598 discloses a method for inhibiting the binding of a β-sheet fibril, such as amyloid fibril to RAGE on the surface of a cell, by using a fragment of RAGE.